During the past years the use of glycolipids as immunostimulating agents has become increasingly important, mainly due to their potential role in the defence against infections, tumour immune surveillance and auto-immunity. An extensively studied subset of T lymphocytes are natural killer T (NKT) cells, which express an invariant T cell antigen receptor (TCR). NKT cells are a subset of regulatory T cells that are involved in different pathological processes, ranging from autoimmunity to protection against tumors and bacterial infections. NKT cell activation results in immediate cytokine production (within several hours), cytotoxicity and proliferation, which subsequently activate several by-stander immune cells (NK cells, dendritic cells, B cells, etc.). Unlike other T cells, NKT cells recognize glycolipid antigens when presented by the major histocompatibility complex (MHC) class I-like molecule CD1d.
The most well studied CD1d-presented antigen that specifically activates invariant NKT (hereinafter iNKT) cells is α-galactosyl ceramide (60 -GalCer, also known as KRN7000 and shown as 1 on top of FIG. 1). It resulted from the structural optimisation of a series of agelasphins, a family of α-linked glycosphingolipids isolated from Agelas mauritianus, a marine sponge from which extracts had demonstrated anti-tumour properties in murine models. α-GalCer consists of a galactosyl moiety α-linked to D-erythro-phytosphingosine, which is N-acylated with a 26-carbons fatty acid.
α-GalCer has been proposed as a promising agent for the treatment of cancer, malaria, hepatitis B, certain bacterial infections and the suppression of auto-immune diseases. Its activity relies on the recognition of the CD1d-α-GalCer bimolecular complex. Upon this recognition, NKT cells are activated, resulting in the rapid release of T helper 1 (Th1) and T helper 2 (Th2) cytokines. Secondary activation of other cell types include NK cells, B cells, CD8+ T cells, dendritic cells, and myeloid cells as well as the differentiation of CD4+ T cells into either Th1 or Th2 cells. This ability to influence both innate and adaptive immune responses puts NKT cells in the position to play a pivotal role in regulating immune responses in both host defence and autoimmune diseases.
Th1 cytokines, such as IFN-γ, are stimuli which drive the development of naive helper T cells toward Th1 type cell formation. In contrast, Th2 cytokines like IL-4 send pre-Th cells down the path of Th2 type cell formation. Th1 cells participate in cell-mediated immunity and are essential for controlling intracellular pathogens, while Th2 cells participate in antibody-mediated immunity control of extra-cellular pathogens. The balance between Th1 and Th2 cytokines is carefully controlled and any disruption between the two can cause disease. Therapeutic strategies could involve trying to restore Th1/Th2 balance through in vivo modulation of NKT cells. While certain auto-immune diseases are characteristic of hypo-responsiveness to Th2 and over-activation of Th1 cells, the opposite is true for many types of cancer that have a predominant Th2 response. Th1 cytokines are thought to mediate the anti-tumour, antiviral, and antibacterial effects of α-GalCer. In a Phase I study, α-GalCer was ineffective in the treatment of solid tumours possibly because the therapeutic effects of IFN-γ were hindered by IL-4 giving no net benefit. Skewing of the cytokine release profile to Th1 would be beneficial for the treatment of these diseases and, therefore, the development of α-GalCer analogues capable to induce a biased Th1 response while maintaining α-GalCer's antigenic potency are highly awaited.
Although initially iNKT cell research was mainly focused on this antigen, the list of novel glycolipids that are able to induce iNKT cell activation is continuously growing and includes very diverse bacterial antigens and endogenously expressed glycolipids, in addition to newly synthesized antigens. Attempts to selectively control the rapid secretion of cytokines by NKT cells have led to the development of several α-GalCer analogues with immunomodulatory properties, as shown on FIG. 10.
The iNKT cell T-cell receptor is semi-invariant as it contains a conserved Vα14 chain in mice and Vα24 in human, while the Vβ chain is more variable. However, only germline encoded residues are important for the recognition of a glycolipid. Although the T-cell receptor plays an important role for initial recognition of the CD1d-glycolipid complex, the strength of a Th1 polarized iNKT cell dependent activation seems to be more determined by the stability of the CD1d-glycolipid complex. Most analogues capable to induce a polarised response reported so far originate from modifications of the hydrophobic chains of α-GalCer. With the synthesis of OCH (structure shown in FIG. 10), an α-GalCer analogue with truncated sphingosine and fatty acyl chains, a direct relationship has been shown relating the shortening of lipid tail lengths and biasing of the cytokine release profile toward a Th2 response. Likewise, it was reported that substituting the N-acyl chain of α-GalCer with shorter, unsaturated fatty acids modifies the outcome of Vα14i NKT cell activation. Analogues containing multiple cis-double bonds in the acyl chain (e.g. shown as 4 in FIG. 10) potently induced a T helper type 2-biased cytokine response, with diminished IFN-γ production and reduced Vα14i NKT cell expansion. Conversely, it has been found that introducing terminal aromatic groups into the fatty acyl tail of α-GalCer (such as in compound 5 of FIG. 10) enhances stability of the glycolipid/CD1d complex and biases the profile toward a Th1 response.
Characterised by an enhanced Th1 response, α-C-GalCer (shown as compound 3 in FIG. 10) exhibits 100- to 1000-fold improved activity against melanoma metastases and malaria compared with α-GalCer. Both are diseases where a Th1 response is beneficial. In this analogue, the O linkage between the sugar and ceramide is replaced with a C-linkage giving the glycosidic bond in vivo stability to enzymatic degradation. It is unclear to what extent the enhanced stability of this compound accounts for its superior in vivo activity. Likewise, a protective effect of OCH was found on Th1-mediated auto-immune diseases, such as collagen-induced arthritis (CIA) and experimental auto-immune encephalomyelitis (EAE) in mice. Recently it was demonstrated that in vivo neutralisation of IFN-γ release induced by α-GalCer early during the course of disease resulted in partial improvement of clinical arthritis symptoms, further indicating the importance of a skewed cytokine profile on the therapeutical outcome.
In 2005, the crystal structure of human CD1d complexed with α-GalCer was elucidated and unravelled the specific binding mode of α-GalCer to CD1 d. The acyl chain of α-GalCer fits into the A′ pocket by adopting a counter-clockwise circular curve, while the sphingosine chain adopts an extended conformation to fit into the F′ pocket and to reach the end of the binding groove. The galactose ring is well ordered and extends above the surface of the lipid-binding groove. The crystal structure revealed three hydrogen bonds between human CD1d and α-GalCer. The glycosidic linkage 1″-O is hydrogen-bonded to Thr-154, the 2″-OH of the galactose ring forms a hydrogen bond to Asp-151 and the 3-OH of the sphingosine moiety forms the third hydrogen bond to Asp-80. These bonds are assumed to anchor α-GalCer in a proper orientation for recognition by the T-cell receptor of NKT cells.
Previously the naphthylurea derivative NU-α-GalCer was shown to induce a structural change within the A′ roof of CD1d to which it binds with its hydrophobic 6″-naphthylurea group, leading to the so called third anchor model. Data suggested that the formation of an extra anchor leads to stronger anti-tumoral responses in vivo. However, the Th1 polarizing strength seemed to be critically dependent on the nature and length of the linker between C-6″ of the galactose and the aromatic groups. BnNH-GSL-1′, an analogue characterized by an aromatic moiety located one atom closer to the galactose ring, was shown to affect T-cell receptor affinity and decreased antigenicity despite the fact that its amide linker, just like the urea linker of NU-α-GalCer, forms an additional H-bond with CD1d.
Extra binding strength of a glycolipid can also be achieved through alterations of the lipid tails. The altered sphingosine chain of a plakoside analogue was shown to increase the contact surface area with CD1d within the F′-pocket. Additionally it was shown that several acyl chain altered glycolipids can induce superior anti-cancer effects compared to α-GalCer and this was also linked to increased CD1d avidity. Last but not least crystallographic analysis of iGb3, a beta-anomeric tri-hexose containing sphingolipid self-antigen, demonstrated that the T-cell receptor was able to bind to the CD1d-glycolipid complex with its conserved footprint. This mechanism induces the last anchor sugar to bind to CD1d, however this does not happen to Gb3, which only differs by an altered linkage of the last sugar, because Gb3 was not able to form this additional anchor to CD1d.
Recently the structure of a human NKT T-cell receptor in complex with CD1d bound to α-GalCer was reported. Consistent with the previously proposed structures, α-GalCer protrudes minimally from the CD1d cleft with only the galactosyl head group exposed for recognition by the NKT T-cell receptor, interacting solely with the CDR1α and CDR3α loops. The galactose ring is sandwiched between Trp-153 of CD1d and the aliphatic moiety of Arg-95α, the side chain of which also hydrogen bonds to the 3-OH on the sphingosine chain. The 2′-OH, 3′-OH and 4′-OH of the galactose ring form hydrogen bonds to Gly-96α, Ser-30α and Phe-29α, respectively, located on the invariant TCR α-chain. This mode of binding is consistent with the specificity the NKT TCR exhibits for α-GalCer and closely related analogues.
The issue of galactosylceramide therapy for auto-immune diseases has been discussed extensively by L. Van Kaer in Nature Reviews (2005) 5:31-42 and references cited therein, the content of which is incorporated herein by reference. Other biological and synthetic issues in respect of certain galactosylceramides have also been disclosed by Kratzer et al in Eur. J. Org. Chem. (1998) 291-298; Zhou et al in Org. Lett. (2002) 4:1267-1270; Yang et al in Angew. Chem. Intl. Ed. (2004) 43:3818-3822; azido and arylurea derivatives of α-GalCer in Trappeniers et al, J. Am. Chem. Soc. (2008) 130:16468-9; and Trappeniers et al in Org. Lett. (2010) 12:2928-2931, the content of which is incorporated herein by reference.
WO 2004/094444 discloses a 6-substituted amino-6-deoxy-galactosylceramide derivative with NKT cell stimulating activity. WO 2007/118234 also discloses a 6-acylamino derivative for staining and stimulating natural killer T cells.
However none of these galactopyranosyl derivatives has been shown to exhibit both the strong biological activity and the safety/release profile that can make them an acceptable drug to be made commercially available for the treatment of diseases.
Accordingly there is still a need in the art for alternative α-GalCer analogues exhibiting enhanced biological activities, enhanced drug formulation capabilities, and improved safety profile.
There is also a regular need in the art for novel compounds acting as anti-cancer agents that can be used, alone or in combination with another form or therapeutic treatment, for treating various forms of cancer.
There is also a regular need in the art for novel compounds having significant and specific anti-parasitic or anti-infectious properties without having the drawbacks of known effective anti-parasitic agents. There is a regular need in the art for effective anti-parasitic or anti-infectious agents having improved metabolisation and/or pharmacokinetic behaviour and which therefore can be more easily formulated into effective dosage forms. There is also a need in the art for such novel compounds exhibiting a longer plasma half-life and a significantly improved resorption rate. There is also a regular need in the art for novel specific and highly therapeutically active compounds having significant immuno-modulating or immunosuppressive activity, such as, but not limited to, drugs for treating immune or autoimmune disorders, and organ and cells transplant rejections